In eukaryotic cells, formation of a bipolar mitotic spindle is essential for stable propagation of the genome. In humans, errors in this process are associated with cancer. Essentially all of the proteins required to organize microtubules into a bipolar spindle are now known. However, the precise functions of most of these proteins and how their functions are coordinated remain mysterious. Eg5 is a widely conserved mitotic motor protein whose loss of function leads to monopolar spindles and mitotic arrest. Chemical inhibitors of Eg5 are currently in clinical trials as cancer therapeutics. Eg5 has been shown to slide microtubules relative to one another, a key microtubule organizing function required to establish the spindle's bipolar geometry. However, how microtubule sliding is coupled to the motility of Eg5 and how this motor protein is regulated remain unknown. To address these outstanding questions, single molecule fluorescence microscopy will be combined with micromanipulation by optical trapping to characterize the motility of individual Eg5 molecules sliding microtubules relative to one another. The involvement of allosteric regulatory mechanisms in Eg5 function will be investigated, and mutated constructs of Eg5 will allow analysis of how Eg5's structural architecture influences its motility and regulation. Finally, development of a new strategy for fluorescent labeling of Eg5 compatible with center-of-mass labeling will increase the accessibility of Eg5 to study by powerful biophysical methods such as FIONA and FRET. The detailed characterization of Eg5 function will provide important insights into the fundamental cellular process of mitotic spindle assembly. PUBLIC HEALTH RELEVANCE. Eg5, a molecular motor in vertebrate animals, is an essential component of the machinery needed for cell division and the distribution of genetic material into new cells. In humans, errors in these processes are associated with developmental defects and cancer. Understanding how this protein works will provide important insights into fundamental principles of cell biology, and will contribute to the development of better cancer prevention and treatments.